Depolymerization of SUMO chains induces slender to stumpy differentiation in T. brucei bloodstream parasites.

Trypanosoma brucei are protozoan parasites that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, a quorum sensing-like mechanism coordinates its differentiation from a slender replicative form into a quiescent stumpy form, limiting growth and activating metabolic pathways that are beneficial to the parasite in the insect host. The post-translational modification of proteins with the Small Ubiquitin-like MOdifier (SUMO) enables dynamic regulation of cellular metabolism. SUMO can be conjugated to its targets as a monomer but can also form oligomeric chains. Here, we have investigated the role of SUMO chains in T. brucei by abolishing the ability of SUMO to polymerize. We have found that parasites able to conjugate only SUMO monomers are primed for differentiation. This was demonstrated for monomorphic lines that are normally unable to produce stumpy forms in response to quorum sensing signaling in mice, and also for pleomorphic cell lines in which stumpy cells were observed at unusually low parasitemia levels. SUMO chain mutants showed a stumpy compatible transcriptional profile and better competence to differentiate into procyclics. Our study indicates that SUMO depolymerization may represent a coordinated signal triggered during stumpy activation program.

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

Cell-to-flagellum attachment and surface architecture in kinetoplastids.

A key morphological feature of kinetoplastid parasites is the position and length of flagellum attachment to the cell body. This lateral attachment is mediated by the flagellum attachment zone (FAZ), a large complex cytoskeletal structure, which is essential for parasite morphogenesis and pathogenicity. Despite the complexity of the FAZ only two transmembrane proteins, FLA1 and FLA1BP, are known to interact and connect the flagellum to the cell body. Across the different kinetoplastid species, each only has a single FLA/FLABP pair, except in Trypanosoma brucei and Trypanosoma congolense where there has been an expansion of these genes. Here, we focus on the selection pressure behind the evolution of the FLA/FLABP proteins and the likely impact this will have on host-parasite interactions.

The Trypanosomatids Cell Cycle: A Brief Report.

Trypanosomatids are protozoan parasites among which are the etiologic agents of various infectious diseases in humans, such as Trypanosoma cruzi (causative agent of Chagas disease), Trypanosoma brucei (causative agent of sleeping sickness), and species of the genus Leishmania (causative agents of leishmaniases). The cell cycle in these organisms presents a sequence of events conserved throughout evolution. However, these parasites also have unique characteristics that confer some peculiarities related to the cell cycle phases. This review compares general and peculiar aspects of the cell cycle in the replicative forms of trypanosomatids. Moreover, a brief discussion about the possible cross-talk between telomeres and the cell cycle is presented. Finally, we intend to open a discussion on how a profound understanding of the cell cycle would facilitate the search for potential targets for developing antiparasitic therapies that could help millions of people worldwide.

Functional characterization of the first lipoyl-relay pathway from a parasitic protozoan.

Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as "lipoyl-relay", requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.

Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana.

BACKGROUND: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. CONCLUSION/SIGNIFICANCE: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

Intrinsically Disordered Region Modulates Ligand Binding in Glutaredoxin 1 from Trypanosoma Brucei.

Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron-sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.

Effect of lysine acetylation on the regulation of Trypanosoma brucei glycosomal aldolase activity.

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.

Measurement of Energy States of the Trypanosomatid Mitochondrion.

The evaluation of mitochondrial functionality is critical to interpret most biological data at the (eukaryotic) cellular level. For example, metabolism, cell cycle, epigenetic regulation, cell death mechanisms, autophagy, differentiation, and response redox imbalance are dependent on the mitochondrial state. In case of parasitic organisms, such as trypanosomatids, it is very often important to have information on mitochondrial functionality in order to assess the mechanisms of actions of drugs being proposed for therapy. In this chapter we present a set of methods that together allow to evaluate with some precision the mitochondrial functionality in Trypanosoma cruzi and Trypanosoma brucei. We discuss how to determine O2 consumption, mitochondrial inner membrane potential, ATP production, and the endogenous production of reactive oxygen species.

Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration.

The co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

Discovery and characterization of trypanocidal cysteine protease inhibitors from the 'malaria box'.

Chagas disease, Human African Trypanosomiasis, and schistosomiasis are neglected parasitic diseases for which new treatments are urgently needed. To identify new chemical leads, we screened the 400 compounds of the Open Access Malaria Box against the cysteine proteases, cruzain (Trypanosoma cruzi), rhodesain (Trypanosoma brucei) and SmCB1 (Schistosoma mansoni), which are therapeutic targets for these diseases. Whereas just three hits were observed for SmCB1, 70 compounds inhibited cruzain or rhodesain by at least 50% at 5 µM. Among those, 15 commercially available compounds were selected for confirmatory assays, given their potency, time-dependent inhibition profile and reported activity against parasites. Additional assays led to the confirmation of four novel classes of cruzain and rhodesain inhibitors, with potency in the low-to mid-micromolar range against enzymes and T. cruzi. Assays against mammalian cathepsins S and B revealed inhibitor selectivity for parasitic proteases. For the two competitive inhibitors identified (compounds 7 and 12), their binding mode was predicted by docking, providing a basis for structure-based optimization efforts. Compound 12 also acted directly against the trypomastigote and the intracellular amastigote forms of T. cruzi at 3 µM. Therefore, through a combination of experimental and computational approaches, we report promising hits for optimization in the development of new trypanocidal drugs.

Trypanosoma brucei Interaction with Host: Mechanism of VSG Release as Target for Drug Discovery for African Trypanosomiasis.

The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.

The Trypanosoma brucei RNA-Binding Protein TbRRM1 is Involved in the Transcription of a Subset of RNA Pol II-Dependent Genes.

It has been long thought that RNA Polymerase (Pol) II transcriptional regulation does not operate in trypanosomes. However, recent reports have suggested that these organisms could regulate RNA Pol II transcription by epigenetic mechanisms. In this paper, we investigated the role of TbRRM1 in transcriptional regulation of RNA Pol II-dependent genes by focusing both in genes located in a particular polycistronic transcription unit (PTU) and in the monocistronic units of the SL-RNA genes. We showed that TbRRM1 is recruited throughout the PTU, with a higher presence on genes than intergenic regions. However, its depletion leads both to the decrease of nascent RNA and to chromatin compaction only of regions located distal to the main transcription start site. These findings suggest that TbRRM1 facilitates the RNA Pol II transcriptional elongation step by collaborating to maintain an open chromatin state in particular regions of the genome. Interestingly, the SL-RNA genes do not recruit TbRRM1 and, after TbRRM1 knockdown, nascent SL-RNAs accumulate while the chromatin state of these regions remains unchanged. Although it was previously suggested that TbRRM1 could regulate RNA Pol II-driven genes, we provide here the first experimental evidence which involves TbRRM1 to transcriptional regulation.

The DNA damage response is developmentally regulated in the African trypanosome.

Genomes are affected by a wide range of damage, which has resulted in the evolution of a number of widely conserved DNA repair pathways. Most of these repair reactions have been described in the African trypanosome Trypanosoma brucei, which is a genetically tractable eukaryotic microbe and important human and animal parasite, but little work has considered how the DNA damage response operates throughout the T. brucei life cycle. Using quantitative PCR we have assessed damage induction and repair in both the nuclear and mitochondrial genomes of the parasite. We show differing kinetics of repair for three forms of DNA damage, and dramatic differences in repair between replicative life cycle forms found in the testse fly midgut and the mammal. We find that mammal-infective T. brucei cells repair oxidative and crosslink-induced DNA damage more efficiently than tsetse-infective cells and, moreover, very distinct patterns of induction and repair of DNA alkylating damage in the two life cycle forms. We also reveal robust repair of DNA lesions in the highly unusual T. brucei mitochondrial genome (the kinetoplast). By examining mutants we show that nuclear alkylation damage is repaired by the concerted action of two repair pathways, and that Rad51 acts in kinetoplast repair. Finally, we correlate repair with cell cycle arrest and cell growth, revealing that induced DNA damage has strikingly differing effects on the two life cycle stages, with distinct timing of alkylation-induced cell cycle arrest and higher levels of damage induced death in mammal-infective cells. Our data reveal that T. brucei regulates the DNA damage response during its life cycle, a capacity that may be shared by many microbial pathogens that exist in variant environments during growth and transmission.

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes.

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise-including whether there is a dedicated and ES-focused mechanism-is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication-transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host.

Nucleolar Division in the Promastigote Stage of Leishmania major Parasite: A Nop56 Point of View.

Nucleogenesis is the cellular event responsible for the formation of the new nucleoli at the end of mitosis. This process depends on the synthesis and processing of ribosomal RNA (rRNA) and, in some eukaryotes, the transfer of nucleolar material contained in prenucleolar bodies (PNBs) to active transcription sites. The lack of a comprehensive description of the nucleolus throughout the cell cycle of the human pathogen Leishmania major prompted us to analyze the distribution of nucleolar protein 56 (Nop56) during interphase and mitosis in the promastigote stage of the parasite. By in silico analysis we show that the orthologue of Nop56 in L. major (LmNop56) contains the three characteristic Nop56 domains and that its predicted three-dimensional structure is also conserved. Fluorescence microscopy observations indicate that the nucleolar localization of LmNop56 is similar, but not identical, to that of the nucleolar protein Elp3b. Notably, unlike other nucleolar proteins, LmNop56 remains associated with the nucleolus in nonproliferative cells. Moreover, epifluorescent images indicate the preservation of the nucleolar structure throughout the closed nuclear division. Experiments performed with the related parasite Trypanosoma brucei show that nucleolar division is carried out by an analogous mechanism.

Simplified inducible system for Trypanosoma brucei.

Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including α-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.

Chromosomal copy number variation analysis by next generation sequencing confirms ploidy stability in Trypanosoma brucei subspecies.

Although aneuploidy usually results in severe abnormalities in multicellular eukaryotes, recent data suggest that it could be beneficial for unicellular eukaryotes, such as yeast and trypanosomatid parasites, providing increased survival under stressful conditions. Among characterized trypanosomatids, Trypanosoma cruzi, Trypanosoma brucei and species from the genus Leishmania stand out due to their importance in public health, infecting around 20 million people worldwide. The presence of aneuploidies in T. cruzi and Leishmania was recently confirmed by analysis based on next generation sequencing (NGS) and fluorescence in situ hybridization, where they have been associated with adaptation during transmission between their insect vectors and mammalian hosts and in promoting drug resistance. Although chromosomal copy number variations (CCNVs) are present in the aforementioned species, PFGE and fluorescence cytophotometry analyses suggest that aneuploidies are absent from T. brucei. A re-evaluation of CCNV in T. b gambiense based on NGS reads confirmed the absence of aneuploidies in this subspecies. However, the presence of aneuploidies in the other two T. brucei subspecies, T. b. brucei and T. b. rhodesiense, has not been evaluated using NGS approaches. In the present work, we tested for aneuploidies in 26 T. brucei isolates, including samples from the three T. brucei subspecies, by both allele frequency and read depth coverage analyses. These analyses showed that none of the T. brucei subspecies presents aneuploidies, which could be related to differences in the mechanisms of DNA replication and recombination in these parasites when compared with Leishmania.

Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation.

Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.

Recruitment kinetics of the homologous recombination pathway in procyclic forms of Trypanosoma brucei after ionizing radiation treatment.

One of the most important mechanisms for repairing double-strand breaks (DSBs) in model eukaryotes is homologous recombination (HR). Although the genes involved in HR have been found in Trypanosoma brucei and studies have identified some of the proteins that participate in this HR pathway, the recruitment kinetics of the HR machinery onto DNA during DSB repair have not been clearly elucidated in this organism. Using immunofluorescence, protein DNA-bound assays, and DNA content analysis, we established the recruitment kinetics of the HR pathway in response to the DSBs generated by ionizing radiation (IR) in procyclic forms of T. brucei. These kinetics involved the phosphorylation of histone H2A and the sequential recruitment of the essential HR players Exo1, RPA, and Rad51. The process of DSB repair took approximately 5.5 hours. We found that DSBs led to a decline in the G2/M phase after IR treatment, concomitant with cell cycle arrest in the G1/S phase. This finding suggests that HR repairs DSBs faster than the other possible DSB repair processes that act during the G1/S transition. Taken together, these data suggest that the interplay between DNA damage detection and HR machinery recruitment is finely coordinated, allowing these parasites to repair DNA rapidly after DSBs during the late S/G2 proficient phases.